ATP UTILIZATION FOR CALCIUM-UPTAKE AND FORCE PRODUCTION IN SKINNED MUSCLE-FIBERS OF XENOPUS-LAEVIS

1. A method has been developed to discriminate between the rate of ATP hydrolysis associated with calcium uptake into the sarcoplasmic retic ulum (SR) and force development of the contractile apparatus in mechan ically or saponin-skinned skeletal muscle fibres. The rate of ATP hydr olysis was determined in fibres of different types from the iliofibula ris muscle of Xenopus laevis by enzymatic coupling of ATP re-synthesis to the oxidation of NADH. 2. The ATPase activity was determined befor e and after exposure of the preparations for 30 min to a solution cont aining 0.5% Triton X-100, which effectively abolishes the SR ATPase ac tivity. The fibres were activated in a solution containing 5 mM caffei ne to ensure that calcium uptake into the SR was maximal. 3. At satura ting Ca2+ concentrations the actomyosin (AM) and SR ATPase activities in fast-twitch fibres, at 4.3 degrees C, amounted to 1.52+/-0.07 and 0 .58+/-0.10 mu mol s(-1) (g dry wt)(-1), respectively (means+/-S.E.M.; n=25). The SR ATPase activity was 25% of the total ATPase activity. At submaximal calcium concentrations the AM ATPase activity varied in pr oportion to the isometric force. 4. The calcium sensitivity of the SR ATPase was larger than that of the AM ATPase and its dependence on [Ca 2+] was less steep. The AM ATPase activity was half-maximal at a pCa o f 6.11 (pCa=-log[Ca2+]) whereas the SR ATPase activity was half-maxima l at a pCa of 6.62. 5. In Triton X-100-treated fibres, at different 2, 3-butanedione monoxime (BDM) concentrations, the AM ATPase activity an d isometric force varied proportionally. The SR ATPase activity determ ined by extrapolation of the total ATPase activity in mechanically ski nned or saponin-treated fibres to zero force, was independent of the B DM concentration in the range studied (0-20 mM). The values obtained f or the SR ATPase activity in this way were similar to those obtained w ith Triton X-100 treatment. 6. The AM ATPase activity in slow-twitch f ibres amounted to 0.74+/-0.13 mu mol s(-1) (g dry wt)(-1), i.e. about a factor of two smaller than in fast-twitch fibres. The SR ATPase acti vity amounted to 0.47+/-0.07 mu mol s(-1) (g dry wt)(-1), i.e. rather similar to the value in fast-twitch fibres. The proportion of the tota l ATPase activity that was due to SR ATPase (40%) was larger than in f ast-twitch fibres. 7. The temperature dependence of the AM and SR ATPa se activities in fast-twitch fibres differed. In the temperature range 5-10 degrees C, the relative changes in AM and SR ATPase activitites for a 10 degrees C temperature change (Q(10)) were 3.9+/-0.3 and 7.2+/ -1.5, respectively. In the temperature range 10-20 degrees C, the Q(10 ) values of the AM and SR ATPase activities were, respectively, 2.6+/- 0.4 and 3.1+/-0.5. As a result the SR ATPase activity at high temperat ure was a larger fraction of the total ATPase activity than at low tem perature.